Hydrogen peroxide is released due to many metabolic reactions in the body, which gives toxicity to the living cells. Hence, there is a chemical need to remove hydrogen peroxide in a very rapid rate. In order to avoid this toxicity, the enzyme Catalase is present in sufficient quantities in the cells. Catalase binds with the substrate molecules though their active sites and catalyses by processing them to form the ultimate by-products of water and oxygen, at a rapid rate as it has high turnover numbers. Every 2 molecules of hydrogen peroxide is broken down into 2 molecules of water and one molecule of oxygen, which are released as the by-products. This is a simple reaction and proceeds as follows:
2 H2O2 >> 2 H2O + O2 (gas)
Michaelis-Menton mechanism in governing the rate of enzyme catalysis:
According to the Michaelis-Menton mechanism, there are three principal features of many enzyme catalysed reactions and they are as follows:
1.For a given initial concentration of substrate, [S]0, the initial rate of product formation is proportional to the total concentration of enzyme [E]0.
2.For a given [E]0 and low values of [S]0, the rate of product formation is proportional to [S]0.
3.For a given [E]0 and high values of [S]0, the rate of product formation becomes independent of [S]0, reaching a maximum value known as the maximum velocity, V(max).
An intensive metabolic activity can be found in soaked pea seeds as they begin to germinate. Hence, their catalytic activity is at higher levels. When a small quantity of hydrogen peroxide is added to it, the enzyme Catalase present in the soaked pea seeds breaks down them into water and oxygen, producing froth due to the release of oxygen gas. This released oxygen can be measured in order to study the enzyme activity.
1.Prepare 6 different concentrations of hydrogen peroxide in gradual increments, taking the standard low concentration of hydrogen peroxide used in the pilot study (as defined in STEP B, POINT 1) as the lowest concentration in the gradually increasing scale.
1.Conduct a pilot study to check acceptable concentrations of hydrogen peroxide for the investigation by measuring the quantity of oxygen evolved from a reaction in which a low concentration of hydrogen peroxide is treated with 3 parts lesser quantity of the enzyme Catalase. Record the quantity of oxygen evolved and during a standard time. The quantities of enzyme and substrate used in this reaction will act as the standard.
2.Crush suitable quantity of pea seeds that will give the standard low concentration of hydrogen peroxide as used in the pilot study, into a coarse powder and load same quantity of the powder into 6 different test tubes (closed test tubes) by crushing more pea seeds as many times as the no. of remaining test tubes.
1.All these reactions must be carried out in a temperature controlled environment, at a constant temperature and pH, as per the requirements of the Catalase enzyme for its reaction with hydrogen peroxide for breaking it into hydrogen and oxygen. This can be accomplished by using a heating block to carry out individual reactions for a standard amount of time.
2.The test tubes used must be of sealed type with a provision for inserting the probe of a suitable oxygen detector into it. This way, the escape of evolved oxygen can be prevented and thus the quantity of oxygen released can be measured more accurately.
3.A stopwatch or any similar device can be used to monitor the time of reaction. All the reactions must be carried out for a standard amount of time.
1.Record the quantity of oxygen evolved in each reaction.
2.Record the concentration and quantity of hydrogen peroxide used in each reaction.
3.The concentration and quantity of enzyme Catalase used in all these reactions is a standard concentration and hence doesn’t vary.
1.Tabulate all the values recorded in the step D.
2.Plot a graph of quantity of oxygen evolved Vs. the concentration of Hydrogen Peroxide.
3.Considering the shape of curve is significant for analysis.
1.The graph will show a steadily rising line, turning into a plateau, after reaching a certain point. The maximal point of steadily raising line is the V(max) / V(M) representing the maximum velocity.
2.In the beginning of the reaction, where the concentration of substrate is high, the Catalase catalyses the reaction more rapidly. The steadily raising line in the beginning of the curve plotted represents the increased rate of catalysis by the enzyme Catalase.
3.After reaching maximum velocity V(max), a plateau region is observed that shows no further increment in the rate of reaction as all of the enzyme molecules are already bounded with substrate molecules. Hence the velocity of reaction remains the same.
1.With this plot, it can be evaluated that:
a.For a given enzyme concentration and low concentration of substrate, the rate of product formation is proportional to the substrate concentration
b.For a given enzyme concentration and high concentration of substrate, the rate of product formation becomes independent of substrate concentration and reaches a maximum velocity known as: V(max) or V(M).
2.Thus, the curve plotted in the STEP E can be interpreted with Michaelis-Menton mechanism of enzyme catalysis for studying the effect of concentration of the substrate.
1.Hydrogen peroxide as a metabolic by-product in the cells.
2.Role of hydrogen peroxide in the cytotoxicity.
3.Role of Catalase in the Red Blood Cells, in prevention of toxicity from hydrogen peroxide.
4.Michaelis-Menton mechanism of enzyme catalysis.
1.Atkins & de Paula, Atkins Physical Chemistry, 7th Edition
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